Hybridization technology is a powerful tool to identify pairs of molecules with a complementary relationship to one another. Such technology has been applied to a variety of types of molecules, including nucleic acids and proteins. For example, nucleic acid hybridization is a well-known and documented method for identifying specific nucleic acid sequences. Nucleic acid hybridization is based on the base pairing of complementary nucleic acid strands. When single stranded nucleic acids are incubated in appropriate buffers, complementary base sequence form double strand stable molecules. The presence or absence of such pairing may be detected by a number of different methods well known in the art. Most hybridization assays previously described involve multiple steps such as the hybridization techniques described by Dunn and Hassell in Cell 12:23 (1977).
The typical hybridization assay protocol for detecting a target nucleic acid in a complex population of nucleic acids can be generally described as follows. Target nucleic acids are separated by size on a gel matrix (electrophoresis), or are cloned and isolated, or sub-divided into pools, or are used in a complex population. The target nucleic acids are then transferred, or spotted, or otherwise immobilized onto a solid support, such as a nylon membrane or nitrocellulose membrane. (This "immobilization" can also be referred to as "arraying.") The immobilized nucleic acids are then subjected to a heating step or UV radiation which irreversibly immobilizes the nucleic acid. The membranes are then immersed in one or more of the traditional "blocking agents" which include Denhardt's reagent (Denhardt, Biochem. Biophys. Res. Comm. 23:641 (1966)), heparin (Singh and Jones, Nucleic Acids Res. 12:5627 (1984)), and non-fat dried milk (Jones et al., Gene Anal. Tech. 1:3 (1984)). Frequently, these reagents are used in combination with single-strand DNA and detergents such as sodium dodecyl sulfate (SDS). In the northern analysis of non-abundant sequences, or hybridizations using RNA probes, or single-copy Southern hybridizations, Denhardt's reagent is generally used with 0.5% SDS and 100 micrograms/ml of fragmented denatured DNA. (50.times. Denhardt's reagent consists of 1% w/v Ficoll (type 400, Pharmacia), 1% w/v polyvinylpyrrolidone, 1% w/v bovine serum albumin (Fraction 5).) Blocking agents are generally included in both the prehybridization step and hybridization steps when nitrocellulose is used. However, when nucleic acid is immobilized on nylon membranes, the blocking agents are generally omitted from the hybridization solution since high concentrations of protein interfere with the annealing of the probe to its target nucleic. The latter problem is particularly noticeable when short probes, such as oligonucleotides, are employed. The target nucleic acids are then typically probed with labelled "signal" nucleic acid under stringent hybridization conditions. Signal nucleic acids are then frequently detected by using a conjugated enzyme in which the conjugated enzyme possesses one member of a ligand pair. The signal nucleic acid contains the other member of the ligand pair. Unbound enzyme is then washed away and the membrane is immersed in a substrate solution. Signal is then detected by colorimetric means, by fluorescence or by chemiluminescence, depending on substrate type. In short form, the hybridization assay protocol can be summarized as follows: Target nucleic acid is immobilized on a solid support. The solid support is treated with blocking agents to prevent spurious binding (non-specific binding; also known as "background") of probes. The solid support is then probed and signal is detected by a variety of means.
The use of a blocking agent to reduce non-specific binding is essential for a number of reasons, including to be able to detect low levels of specific binding which would be masked by the non-specific binding if the latter is not blocked. Unfortunately, the use of traditional blocking agents, such as those described above, has never been a reproducible method. Although tomes have been written on the subject, it is virtually impossible to uniformly "block," for example, an 8 inch by 12 inch piece of nitrocellulose or nylon membrane using a combination or cocktail of blocking reagents composed of Ficoll (type 400, Pharmacia), polyvinylpyrrolidone, bovine serum albumin (Fraction 5, Sigma), fragmented single nucleic acid, milk products, etc. The methods of the present invention as described herein overcome this limitation of previous methods.
Due to the problems associated with the current approaches for reducing non-specific background in hybridization reactions, there is a need in the art for new methods. The present invention fulfills this need, and further provides other related advantages.